Gene expression profi ling of acute myeloid leukemia

Hematopoïese, of de vorming van functionele bloedcellen, is een proces wat plaats vindt in het beenmerg. Hematopoïetische stamcellen ondergaan cycli van deling en differentiatie waarin de functionele eindcellen, zoals rode bloedcellen, bloedplaatjes en witte bloedcellen, worden gevormd. Leukemie is een ziekte waarbij de stamcellen abnormale processen van deling in combinatie met een stop van de differentiatie ondergaan, waardoor er de vorming van functionele eindcellen wordt belemmerd. In het geval van acute myeloïde leukemie (AML) is er een afwijking in de tak van bloedcelvorming waar onder andere rode bloedcellen, bloedplaatjes en granulocyten worden gevormd. De ontsporing van hematopoïetische stamcellen met AML als gevolg wordt veroorzaakt door abnormaliteiten in het genoom, zoals chromosomale fusies, deleties en mutaties. De klinische prognose wordt momenteel bepaald aan de hand van de aan- of afwezigheid van (combinaties van) abnormaliteiten. Het belangrijkste gevolg van genomische afwijkingen is de abnormale transcriptie van genen naar mRNA. Met behulpvan gen expressie profilering, door middel van microarrays, kunnen de transcriptie niveaus van duizenden genen simultaan worden bepaald. In hoofdstuk 2 is een onderzoek beschreven waarin met gen expressie profilering is toegepast op 285 beenmerg monsters van de novo AML patiënten, voor het bepalen van prognose. Verschillende bekende prognostische groepen, zoals t(8;21) en inv(16) konden worden geidentificeerd, alsmede een nieuwe prognostisch relevante groep van patiënten met een relatief slechte prognose (cluster 10).Hoofdstuk 2 laat zien dat gen expressie profilering in staat is om de huidige technieken voor het bepalen van prognose te vervangen, en prognose te verbeteren.Roeland George Willehad Verhaak was born in Wijchen, the Netherlands, on September 29 1976. After fi nishing his VWO education at the Kottenpark College in Enschede in 1996, he started a curriculum Biomedical Health Sciences at the Catholic University Nijmegen (KUN, currently Radboud University). As part of this education, he followed majors in pathobiology and toxicology, and a minor in computer science. A toxicology internship, titled ‘Mitochondrial toxicity of nuclease reverse transcriptase inhibitors, was completed at the Department of Pharmacology and Toxicology of the KUN under supervision of Dr. Roos Masereeuw. A second intership project, ‘Development of a diagnostic marker of multiple sclerosis’, was completed at the Department of Biochemistry, under supervision of Dr. Rinie van Boekel en Prof.dr. W. Van Venrooij. He obtained his Masters–degree in August 2000. After having started a project at the Department of Medical Informatics of the KUN in October 2000 in which he worked on structuring of temporal data, he switched to the bioinformatics company Dalicon BV in April 2002. At Dalicon, he worked as software engineer, with a particular focus at the database system SRS. In April 2003 he started a PhD-project at the Department of Hematology at the Erasmus MC in the lab of Prof.dr. Bob Löwenberg, supervised by Dr. Peter Valk. This work has been described in this thesis. From March 2006 until June 2006, he was a visiting scientist of the Department of Biostatistics and Computational Biology of the Dana-Farber Cancer Institute in Boston, supervised by Prof.dr. John Quackenbush. The author wil continue his academic career at the Broad Institute in Boston, a research collaboration of MIT, Harvard and its affiliated hospitals, and the Whitehead Institute

Prognostically useful gene-expression profiles in acute myeloid leukemia

BACKGROUND: In patients with acute myeloid leukemia (AML) a combination of methods must be used to classify the disease, make therapeutic decisions, and determine the prognosis. However, this combined approach provides correct therapeutic and prognostic information in only 50 percent of cases. METHODS: We determined the gene-expression profiles in samples of peripheral blood or bone marrow from 285 patients with AML using Affymetrix U133A GeneChips containing approximately 13,000 unique genes or expression-signature tags. Data analyses were carried out with Omniviz, significance analysis of microarrays, and prediction analysis of microarrays software. Statistical analyses were performed to determine the prognostic significance of cases of AML with specific molecular signatures. RESULTS: Unsupervised cluster analyses identified 16 groups of patients with AML on the basis of molecular signatures. We identified the genes that defined these clusters and determined the minimal numbers of genes needed to identify prognostically important clusters with a high degree of accuracy. The clustering was driven by the presence of chromosomal lesions (e.g., t(8;21), t(15;17), and inv(16)), particular genetic mutations (CEBPA), and abnormal oncogene expression (EVI1). We identified several novel clusters, some consisting of specimens with normal karyotypes. A unique cluster with a distinctive gene-expression signature included cases of AML with a poor treatment outcome. CONCLUSIONS: Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis

The epigenetic evolution of glioma is determined by the IDH1 mutation status and treatment regimen

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histological progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution towards an IDHwt-like phenotype

Extrachromosomal DNA (ecDNA): an origin of tumor heterogeneity, genomic remodeling, and drug resistance

The genome of cancer cells contains circular extrachromosomal DNA (ecDNA) elements not found in normal cells. Analysis of clinical samples reveal they are common in most cancers and their presence indicates poor prognosis. They often contain enhancers and driver oncogenes that are highly expressed. The circular ecDNA topology leads to an open chromatin conformation and generates new gene regulatory interactions, including with distal enhancers. The absence of centromeres leads to random distribution of ecDNAs during cell division and genes encoded on them are transmitted in a non-mendelian manner. ecDNA can integrate into and exit from chromosomal DNA. The numbers of specific ecDNAs can change in response to treatment. This dynamic ability to remodel the cancer genome challenges long-standing fundamentals, providing new insights into tumor heterogeneity, cancer genome remodeling, and drug resistance

The effect of oligonucleotide microarray data pre-processing on the analysis of patient-cohort studies

Electrical Engineering, Mathematics and Computer Scienc

Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling

We examined the gene expression profiles of two independent cohorts of patients with acute myeloid leukemia [n=247 and n=214 (younger than or equal to 60 years)] to study the applicability of gene expression profiling as a single assay in prediction of acute myeloid leukemia-specific molecular subtypes. The favorable cytogenetic acute myeloid leukemia subtypes, i.e., acute myeloid leukemia with t(8;21), t(15;17) or inv(16), were predicted with maximum accuracy (positive and negative predictive value: 100%). Mutations in NPM1 and CEBPA were predicted less accurately (positive predictive value: 66% and 100%, and negative predictive value: 99% and 97% respectively). Various other characteristic molecular acute myeloid leukemia subtypes, i.e., mutant FLT3 and RAS, abnormalities involving 11q23, -5/5q-, -7/7q-, abnormalities involving 3q (abn3q) and t(9;22), could not be correctly predicted using gene expression profiling. In conclusion, gene expression profiling allows accurate prediction of certain acute myeloid leukemia subtypes, e.g. those characterized by expression of chimeric transcription factors. However, detection of mutations affecting signaling molecules and numerical abnormalities still requires alternative molecular methods

Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): Association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance

Mutations in nucleophosmin NPM1 are the most frequent acquired molecular abnormalities in acute myeloid leukemia (AML). We determined the NPM1 mutation status in a clinically and molecularly well-characterized patient cohort of 275 patients with newly diagnosed AML by denaturing high-performance liquid chromatography (dHPLC). We show that NPM1 mutations are significantly underrepresented in patients younger than 35 years. NPM1 mutations positively correlate with AML with high white blood cell counts, normal karyotypes, and fms-like tyrosine kinase-3 gene (FLT3) internal tandem duplication (ITD) mutations. NPM1 mutations associate inversely with the occurrence of CCAAT/enhancer-binding protein-α (CEBPA) and NRAS mutations. With respect to gene expression profiling, we show that AML cases with an NPM1 mutation cluster in specific subtypes of AML with previously established gene expression signatures, are highly associated with a homeobox gene-specific expression signature, and can be predicted with high accuracy. We demonstrate that patients with intermediate cytogenetic risk AML without FLT3 ITD mutations but with NPM1 mutations have a significantly better overall survival (OS) and eventfree survival (EFS) than those without NPM1 mutations. Finally, in multivariable analysis NPM1 mutations express independent favorable prognostic value with regard to OS, EFS, and disease-free survival (DFS)

Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells

Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to loss of leukemic self-renewal capacity and prevented leukemia in NOD-SCID mice into which leukemic PU.1-knockdown cells were transplanted. Examination of human individuals with AML confirmed the correlation between PU.1 and JunB downregulation. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1-knockdown HSCs and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. Therefore, examination of disturbed gene expression in HSCs can identify genes whose dysregulation is essential for leukemic stem cell function and that are targets for therapeutic interventions

Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia

Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Next-generation sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP3